Supplementary Information for:
Comparative methylome analysis identifies new tumour subtypes and biomarkers for
transformation of nephrogenic rests into Wilms tumour
Jocelyn Charlton1, Richard D. Williams1, Neil J. Sebire1, Sergey Popov2, Gordan Vujanic4, Tasnim Chagtai1,
Mariana Maschietto1, Marisa Alcaide-German1, Tiffany Morris3, Lee M. Butcher3, Paul Guilhamon3, Stephan
Beck3,*, Kathy Pritchard-Jones1,*
1
Figure S1: Validation of the 450k array using bisulfite-sequencing
The β-values discerned by the 450k array (x-axis) compared to level of methylation detected using bisulfitesequencing (y-axis) show good correlation (Pearson correlation coefficient = 0.8365).
2
Figure S2: Probe-wise variance between groups shows NR and WT are much more variable than NK
Variance within the NK group was consistently lower compared to NR and WT groups. Blue bars show observed
data, while the red line indicates the null distribution. We performed Bartlett tests to identify probes showing
significant (p < 0.01) departures from homogeneity of variance between groups. Each time we found the NK
group was substantially skewed toward decreased variability compared with NR groups (skew = -87.89, p <
2.2x10-16, D'Agostino skewness test; Fig. S3a) and WT groups (skew = -160.10, p < 2.2x10-16, D'Agostino
skewness test; Fig. S3b).
a
b
3
Figure S3: Unsupervised analysis shows two Wilms tumour groups
Re-evaluation of tumour separation by multidimensional scaling of the top 1% most variable positions showed
separation of the two Wilms tumour groups 1 (orange) and 2 (red). Group 2 are more intermixed with the NR
samples whereas group 1 are located further away although wide variance is seen.
4
Figure S4: Histological composition of microdissected tissue
Proportions of each cell type (blastema (navy), stroma (green), epithelia (pink) or necrosis/chemotherapy induced
changes (CIC; yellow)) within each microdissected nephrogenic rest (NR) or Wilms tumour (WT) section.
Samples are ordered by patient with pairs labelled with matching letters.
5
Table S3: Description of hypermethylated and hypomethylated differentially methylated regions (DMRs)
in group-1 Wilms tumour compared to matched nephrogenic rests.
Hyper-methylated
Hypo-methylated
DMRs
DMRs
527
1555.5
1130.4
1749.8
108-5,118
110-6,780
186,507
804,907
% enrichment: CG island
0.8
-22.3
% enrichment: CG shore
5.5
-0.5
% enrichment: CG shelf
-2.7
2.2
% enrichment: 'other'
-3.6
20.6
% enrichment: gene body
-3.5
14.6
% enrichment: TSS200
1.6
-5.6
% enrichment: TSS1500
-3.5
-10.4
% enrichment: 3’ UTR
-2.7
-1.3
% enrichment: 5’ UTR
-1.6
-1.6
-1.7
-3.0
11.5
7.2
Median size (bp)
Mean size (bp)
Range (bp)
Total (bp)
% enrichment:
1st
exon
% enrichment: intergenic region
Genetic features and those relating to CpG islands , shores (within 2kb of islands) and shelves (2-4kb from CpG islands) are
extracted from the Illumina 450k annotation. Chromatin data (bivalent, H3K4me3 and H3K27me3 domains) refer to embryonic
stem cells (ESCs) 1.
6
Table S4: Significantly overrepresented biological processes identified by hypomethylated WT-DMRs
Hypergeometric
fold enrichment
Raw pvalue
Axon ensheathment
2.5
1.87E-07
Body morphogenesis
3.04
1.56E-08
Cell chemotaxis
3.27
6.35E-13
Embryonic placenta development
2.74
6.61E-10
Fat cell differentiation
3.16
4.42E-14
Head development
2.77
1.44E-07
Head morphogenesis
3.54
3.44E-10
Intermediate filament-based process
5.44
4.39E-22
Labyrinthine layer development
4.07
9.57E-15
Metanephric nephron development
3.83
2.35E-11
Myelination
2.57
9.16E-08
Myelination in peripheral nervous system
4.65
1.18E-09
Negative regulation of myeloid leukocyte differentiation
6.43
1.48E-22
Nephron development
2.61
1.28E-07
Organ regeneration
3.86
1.14E-13
Palate development
2.8
2.45E-12
Peripheral nervous system development
2.05
6.43E-05
Placenta development
2.42
2.43E-09
Regeneration
2.39
2.63E-08
Regulation of action potential in neuron
2.19
4.73E-06
2
5.30E-14
Regulation of myeloid cell differentiation
2.69
3.62E-12
Regulation of myeloid leukocyte differentiation
3.64
2.07E-14
Regulation of purine nucleotide catabolic process
2.07
1.01E-16
Regulation of Ras GTPase activity
2.14
1.27E-10
Regulation of Ras protein signal transduction
2.02
7.60E-14
Schwann cell development
4.42
3.10E-09
Schwann cell differentiation
3.91
3.11E-08
Stem cell development
2.52
2.46E-09
Stem cell differentiation
2.03
2.01E-06
Stem cell maintenance
2.78
8.61E-11
Enriched Term in GO biological processes
Regulation of GTP catabolic process
7
Table S5: Significantly overrepresented biological processes identified by hypermethylated group 2 WTDMRs
Hypergeometric
fold enrichment
Raw pvalue
Cell adhesion
2.17
3.45E-16
Cell-cell adhesion
4.83
3.00E-44
Homophilic cell adhesion
12.97
5.85E-81
Intracellular receptor mediated signalling pathway
8.07
2.23E-61
Negative regulation of biosyntheic process
2.35
5.46E-30
Negative regulation of cellular biosynthetic process
2.37
1.50E-30
Negative regulation of cellular macromolecule biosynthetic process
2.51
1.22E-33
Negative regulation of cellular metabolic process
2.13
3.76E-26
Negative regulation of gene expression
2.58
1.79E-36
Negative regulation of macromolecule biosynthetic process
2.44
3.08E-32
Negative regulation of macromolecule metabolic process
2.1
2.59E-25
Negative regulation of metabolic process
2.03
6.12E-24
Negative regulation of nitrogen compound metabolic process
Negative regulation of nucleobase-containing compound metabolic
process
2.5
1.70E-33
2.51
1.04E-33
Negative regulation of protein metabolic process
4.19
1.92E-31
Negative regulation of RNA metabolic process
2.61
5.53E-36
Negative regulation of transcription from RNA polymerase II promoter
3.79
8.10E-54
Negative regulation of transcription, DNA-dependent
2.67
2.24E-37
Positive regulation of gene expression
2.06
4.97E-22
Positive regulation of RNA metabolic process
2.06
1.76E-21
Positive regulation of transcription from RNA polymerase II promoter
2.87
4.43E-35
Positive regulation of transcription, DNA-dependent
2.13
4.89E-23
Regulation of gene expression, epigenetic
5.98
2.39E-30
Regulation of organelle organisation
3.57
3.43E-34
Regulation of transcription from RNA polymerase II promoter
2.41
9.04E-40
Transcription from RNA polymerase II promoter
4.87
5.31E-49
Enriched Term in GO biological processes
8
Table S6: Differentially expressed genes in WT compared to NR
Ensembl ID
ENSG00000134917
ENSG00000091972
ENSG00000125734
ENSG00000110492
ENSG00000162601
ENSG00000149294
ENSG00000109320
ENSG00000138078
ENSG00000134198
Gene symbol
ADAMTS8
CD200
GPR108
MDK
MYSM1
NCAM1
NFKB1
PREPL
TSPAN2
P-val (adjusted)
0.01781
1.03E-09
0.01781
0.0008816
0.01284
0.01781
0.02555
3.68E-06
0.005864
Average reads NR
52.67
317.67
46.00
1.00
50.67
6.67
49.67
164.67
15.67
Average reads WT
3.00
3.00
1.75
29.50
3.75
57.25
3.50
4.50
0.00
9
Table S7: Description of hypermethylated and hypomethylated differentially methylated regions (DMRs)
in nephrogenic rests compared to matched normal kidney
Hyper-methylated
Hypo-methylated
DMRs
DMRs
646
1513.5
1418.5
1701.7
104-8,088
154-5,807
487,962
483,284
% enrichment: CG island
-11.5
-21.9
% enrichment: CG shore
9.9
2.5
% enrichment: CG shelf
-1.0
4.1
% enrichment: 'other'
2.5
15.3
% enrichment: ESCs bivalent domain
10.8
-1.9
% enrichment: ESCs H3K4me3 only
-3.0
-15.1
% enrichment: ESCs H3K27me3 only
3.6
1.7
% enrichment: ESCs no H3K27 or H3K4-me3
-11.4
15.3
% enrichment: gene body
-1.3
16.5
% enrichment: TSS200
-2.1
-5.6
% enrichment: TSS1500
-3.3
-6.2
% enrichment: 3’ UTR
1.0
-0.3
% enrichment: 5’ UTR
0.0
-0.8
-2.0
-2.4
7.7
-1.2
Median size (bp)
Mean size (bp)
Range (bp)
Total (bp)
% enrichment:
1st
exon
% enrichment: intergenic region
Genetic features and those relating to CpG islands , shores (within 2kb of islands) and shelves (2-4kb from CpG islands) are
extracted from the Illumina 450k annotation. Chromatin data (bivalent, H3K4me3 and H3K27me3 domains) refer to embryonic
stem cells (ESCs) 1.
10
Table S8: Significantly overrepresented biological processes identified by hypomethylated KR-DMRs
Hypergeometric
fold enrichment
Raw pvalue
Actin cytoskeleton organisation
2.43
1.31E-13
Actin filamentbased process
2.26
5.07E-12
Chromosome organisation involved in meiosis
10.76
1.33E-18
Collagen fibril organisation
6.17
3.30E-19
Extracellular matrix organisation
2.28
2.23E-07
Fat cell differentiation
4.94
4.72E-24
Hormone biosynthetic process
4.43
1.72E-13
Hormone metabolic process
2.52
7.31E-08
Induction of apoptosis
2.24
1.70E-13
Induction of programmed cell death
2.22
2.56E-13
Meiosis
2.75
3.61E-07
Meiosis I
4.43
6.91E-11
Meiotic cell cycle
Negative regulation of transforming growth factor beta receptor
signalling pathway
Negative regulation of transmembrane receptor protein
serine/threonine kinase signalling pathway
2.71
4.72E-07
6.38
2.22E-23
4.47
3.18E-17
Positive regulation of GTPase activity
2.48
2.46E-15
Positive regulation of Ras GTPase activity
3.31
1.89E-16
Regulation of GTP catabolic process
2.28
1.75E-14
Regulation of GTPase activity
2.25
1.39E-13
Regulation of myeloid cell differentiation
3.14
4.19E-12
Regulation of myeloid leukocyte differentiation
5.43
2.51E-21
Regulation of Ras GTPase activity
2.63
5.98E-13
Regulation of Ras protein signal transduction
Regulation of transforming growth factor beta receptor signalling
pathway
Regulation of transmembrane receptor protein serine/threonine kinase
signalling pathway
2.14
1.12E-11
3.99
2.28E-15
2.7
1.29E-11
Synapsis
11.04
7.12E-19
Synaptonemal complex assembly
16.25
5.36E-23
Synaptonemal complex organisation
13.37
6.78E-21
White fat cell differentiation
13.41
1.05E-42
Enriched Term in GO biological processes
11
Table S9: Significantly overrepresented biological processes identified by hypermethylated KR-DMRs
Hypergeometric
fold enrichment
Raw pvalue
Amine transport
2.99
9.88E-15
Anterior/posterior axis specification
2.5
1.16E-04
Anterior/posterior axis specification, embryo
5.39
4.04E-10
Anterior/posterior pattern specification
2.13
9.87E-11
Axis specification
2.24
2.78E-06
Blastoderm segmentation
4.98
1.36E-10
Cell-cell adhesion
2.08
2.92E-11
Cell-cell signalling involved in cell fate commitment
3.73
1.16E-08
Developmental induction
4.05
2.12E-09
Embryonic axis specification
3.27
1.28E-06
Embryonic organ morphogenesis
2.05
1.18E-10
Embryonic pattern specification
2.17
1.29E-04
Homophilic cell adhesion
4.91
1.09E-29
Intracellular receptor-mediated signalling pathway
3.57
7.02E-25
Negative regulation of kinase activity
3.39
7.73E-23
Negative regulation of organelle organisation
2.94
1.40E-15
Negative regulation of protein kinase activity
3.75
9.52E-26
Negative regulation of protein modification process
2.52
3.46E-11
Negative regulation of transferase activity
3.35
6.67E-24
Nitrogen compound transport
3.22
2.34E-19
Peptidyl-proline modification
4.17
5.81E-10
Protein peptidyl-prolyl isomerisation
4.6
6.36E-11
Regulation of adaptive immune response based on somatic
recombination of immune receptors built from immunoglobulin
superfamily domains
2.02
3.71E-03
Regulation of T-helper 1 type immune response
7.62
2.07E-11
Transition metal ion transport
3.55
9.90E-10
Enriched Term in GO biological processes
12
References
1
Pan, G. et al. Whole-Genome Analysis of Histone H3 Lysine 4 and Lysine 27 Methylation in
Human Embryonic Stem Cells. Cell Stem Cell 1, 299-312,
doi:http://dx.doi.org/10.1016/j.stem.2007.08.003 (2007).
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